Highly Active Antiretroviral Therapy (HAART) greatly reduces quantity of active virus and has resulted in a marked increase in life expectancy of HIV-infected (HIV+) individuals. While HAART has also led to a reduction in the incidence of AIDS-defining illnesses, a variety of HIV-Associated Non-AIDS (HANA) conditions more commonly associated with older age are increasingly commonplace in HIV+ individuals. Aging and HIV appear to share common features of immune dysfunction, and the two may act synergistically in the aging population with long term HIV infection. A burgeoning literature suggests that HIV infection accelerates aging; however the underlying molecular mechanisms remain poorly understood. In particular, it not known how measures of disease severity, such as HIV viral load, affect dynamic epigenetic processes such as DNA methylation (DNAm). It is also not known how long term use of HAART impacts DNAm. The PI recently developed a novel biomarker of aging (referred to as epigenetic clock), which is the first age prediction method based on DNAm levels that accurately predicts age in more than one human tissue or fluid. As a matter of fact, it works in the vast majority of tissues/fluids/organs. It is arguably the first accurate measure of age that allows one to compare the ages of different parts of the human body. Using two DNAm data sets from the peripheral blood mononuclear cells of HIV+ individuals, we have demonstrated that even low levels of HIV replication significantly accelerates age according to the epigenetic clock. This R21 proposal will generate crucial data for testing the plausible hypothesis that HIV viral load also accelerates aging in a wide variety of affected human tissues, thus helping to explain the higher incidence of HANA conditions affecting a variety of organ systems. The genome wide DNAm profiles will not only allow us to test this hypothesis but more broadly allow us to identify additional DNAm markers that correlate with viral load in different tissues. To study the clinical ramifications, we will correlate the methylation data with various markers of tissue pathology typically associated with aging (e.g., fibrosis), and pre-mortem diagnoses of HANA conditions. To study the effect of antiretroviral medication (ARV), we will correlate the methylation data with ARV usage data tracked up to 15 years pre-mortem while participants were enrolled in a longitudinal cohort study. This proposal leverages the tissue samples from deceased HIV+ and HIV- subjects and the latest version of the well-validated Ilumina Infinium 450K array, allowing high-resolution genome wide DNAm profiles. Our overarching goal is to show that accelerated cellular aging, as measured by DNAm, has clinical relevance in the context of HIV/HANA. Working from that foundation, we will further investigate the molecular mechanisms in in vitro systems. This research will lead to development of epigenetic biomarkers of HANA conditions, produce useful surrogate measures of clinical outcomes, and open up a new area for therapeutics.